本文選題：玉米 + NAC膜結(jié)合轉(zhuǎn)錄因子��； 參考：《山東農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：轉(zhuǎn)錄因子在調(diào)控植物發(fā)育以及抗逆功能基因的表達(dá)中具有重要作用,其中,NAC轉(zhuǎn)錄因子是植物特有的轉(zhuǎn)錄調(diào)控因子,在植物的器官發(fā)育、形態(tài)建成、器官分離、葉片衰老以及非生物脅迫響應(yīng)中發(fā)揮重要的調(diào)控作用。膜結(jié)合轉(zhuǎn)錄因子是一類特殊的轉(zhuǎn)錄因子,通常情況下,它以膜結(jié)合的形式存在于細(xì)胞的膜結(jié)構(gòu)(細(xì)胞膜、內(nèi)質(zhì)網(wǎng)膜或核膜)上,處于非活性狀態(tài)。在受到外界信號(hào)刺激后,通過(guò)蛋白水解機(jī)制從膜上釋放下來(lái),轉(zhuǎn)變?yōu)橛谢钚缘男问竭M(jìn)入細(xì)胞核發(fā)揮作用。目前,有關(guān)玉米NAC膜結(jié)合轉(zhuǎn)錄因子(ZmNTLs)的功能研究尚未見(jiàn)報(bào)道。本研究從玉米基因組中,發(fā)掘出了7個(gè)ZmNTLs基因,對(duì)它們的蛋白結(jié)構(gòu)特點(diǎn)、系統(tǒng)進(jìn)化樹(shù)、亞細(xì)胞定位、轉(zhuǎn)錄激活活性、組織特異性表達(dá)及其對(duì)非生物脅迫及激素響應(yīng)特性進(jìn)行了研究,獲得了擬南芥35S::ZmNTL1、35S::ZmNTL2和35S::ZmNTL5純系,并分別研究了ZmNTL1,-2在調(diào)控?cái)M南芥水淹脅迫響應(yīng)和木質(zhì)素合成中的作用。主要研究過(guò)程及實(shí)驗(yàn)結(jié)果包括:(1)玉米NAC膜結(jié)合轉(zhuǎn)錄因子ZmNTLs序列、表達(dá)特征分析利用生物信息學(xué)方法,從216個(gè)玉米NAC轉(zhuǎn)錄因子中篩選出7個(gè)具有跨膜結(jié)構(gòu)域的成員,確定為玉米NAC膜結(jié)合轉(zhuǎn)錄因子(ZmNTL),并命名為ZmNTL1-7。序列比對(duì)發(fā)現(xiàn),所有的玉米NTLs N-末端均具有典型的NAC結(jié)構(gòu)域,C-末端均具有1個(gè)跨膜螺旋結(jié)構(gòu)域。氨基酸序列比對(duì),將7個(gè)ZmNTLs分成4個(gè)組(包括3個(gè)基因?qū)?,分別為ZmNTL1/5、ZmNTL2/3、ZmNTL6/7和ZmNTL4。(2)ZmNTLs進(jìn)化分析利用MEGA6.0對(duì)來(lái)自擬南芥的18個(gè)NTLs蛋白、水稻的5個(gè)NTLs蛋白、谷子的8個(gè)NTLs蛋白同玉米的7個(gè)NTL蛋白進(jìn)行了進(jìn)化樹(shù)構(gòu)建,結(jié)果表明上述38個(gè)膜結(jié)合轉(zhuǎn)錄因子被分成5個(gè)亞組,ZmNTLs分布于其中的I-IV 4個(gè)亞組中,ZmNTL2/ZmNTL3和At NTL4位于第I組內(nèi),ZmNTL4和At NTL7位于第II組內(nèi),ZmNTL1/ZmNTL5、At NTL8和At NTL5位于第III組內(nèi),ZmNTL6/ZmNTL7和At NTL6和At NTL9位于第IV組內(nèi),預(yù)測(cè)在一個(gè)亞組中的成員可能有相似功能。通過(guò)MEME進(jìn)行的保守motif分析表明,玉米NTLs的保守motif比擬南芥多。ZmNTLs與At NACs、Os NACs的進(jìn)化關(guān)系分析發(fā)現(xiàn),ZmNTLs與擬南芥中的某些NAC轉(zhuǎn)錄因子具有較近的進(jìn)化關(guān)系,例如ZmNTL1與ANAC086、ANAC045,ZmNTL2與VND6、VND7等,這預(yù)示著ZmNTLs可能與某些擬南芥的NAC有相似的功能。(3)ZmNTLs亞細(xì)胞定位及轉(zhuǎn)錄激活活性分析以玉米B73自交系為材料,克隆了7個(gè)ZmNTLs基因的CDS,構(gòu)建了它們的GFP瞬時(shí)表達(dá)載體,將它們轉(zhuǎn)化到擬南芥原生質(zhì)體中,發(fā)現(xiàn)ZmNTL1-5和7定位于細(xì)胞質(zhì)膜上;ZmNTL6定位于內(nèi)質(zhì)網(wǎng)膜上。酵母轉(zhuǎn)錄激活活性分析發(fā)現(xiàn),所有的ZmNTLs均有轉(zhuǎn)錄激活活性。β-半乳糖苷酶報(bào)告基因定量檢測(cè)表明ZmNTL2轉(zhuǎn)錄激活活性最高,其次是ZmNTL6;ZmNTL4與ZmNTL7相對(duì)較弱。(4)ZmNTLs的時(shí)空表達(dá)模式Real-time PCR分析表明,ZmNTL1-7表達(dá)具有明顯的組織特異性。ZmNTL1,-2在根、莖、葉中均有表達(dá),其中ZmNTL1在葉中表達(dá)量最高,ZmNTL2在莖中表達(dá)量最高,ZmNTL3-7主要在莖中表達(dá)。(5)ZmNTLs對(duì)非生物脅迫及激素的響應(yīng)模式對(duì)三葉期玉米幼苗進(jìn)行了非生物脅迫(干旱、高鹽、低溫、H2O2)及激素(ABA、BR、GA、2,4-D、Me JA、KT)處理下的Real-time PCR分析,結(jié)果表明ZmNTL1-7對(duì)非生物脅迫及激素響應(yīng)的表達(dá)模式表現(xiàn)出多樣性特征。在ABA、H2O2脅迫下,ZmNTL1-7在根和莖中均顯著上調(diào),表達(dá)模式相近;而在PEG處理下,ZmNTL1-7在莖中表達(dá)顯著下調(diào);ZmNTL1-7均受ABA、Me JA、BR、2,4-D、KT誘導(dǎo)為上調(diào)表達(dá);尤其對(duì)ABA和Me JA響應(yīng)最為強(qiáng)烈;GA處理下表達(dá)最弱,ZmNTL1,-4,-6有少量誘導(dǎo),ZmNTL7被抑制,ZmNTL2,-3,-5沒(méi)有明顯變化。(6)玉米ZmNTL1、ZmNTL2、ZmNTL5擬南芥過(guò)表達(dá)系的獲得將ZmNTL1、ZmNTL2、ZmNTL5全長(zhǎng)及其去除跨膜結(jié)構(gòu)域(△TM)的片段連接到過(guò)表達(dá)載體p K2GW7中,分別獲得了它們的轉(zhuǎn)基因擬南芥純系種子。發(fā)現(xiàn)與在正常生長(zhǎng)條件下,它們的主根長(zhǎng)度及苗生長(zhǎng)勢(shì)與野生型無(wú)差異。(7)ZmNTL擬南芥過(guò)表達(dá)系對(duì)過(guò)氧化氫脅迫的響應(yīng)ZmNTL1、-2、-5去除跨膜結(jié)構(gòu)域(△TM)的轉(zhuǎn)基因擬南芥過(guò)表達(dá)系野生型(Col-0)的種子分別點(diǎn)在1/2MS固體培養(yǎng)基上,根長(zhǎng)到1cm左右時(shí),轉(zhuǎn)移到含有0.5mM、1mM、1.5mM和2mM H2O2的培養(yǎng)基上進(jìn)行脅迫處理,結(jié)果表明轉(zhuǎn)基因系能夠明顯緩解了過(guò)氧化氫的抑制作用,這三個(gè)ZmNTLs基因都響應(yīng)過(guò)氧化氫脅迫。(8)ZmNTL1在水淹脅迫中的作用研究在土壤水分飽和的狀態(tài)下,35S::ZmNTL1轉(zhuǎn)基因擬南芥生長(zhǎng)狀態(tài)與對(duì)照無(wú)明顯差異,35S::ZmNTL1-△TM生長(zhǎng)狀態(tài)明顯弱于對(duì)照,表現(xiàn)出發(fā)育遲緩、植株矮化、葉片卷曲,表明ZmNTL1降低了擬南芥對(duì)水淹脅迫的耐受性。Real-time PCR分析表明,在水淹下,35S::ZmNTL1-△TM中的At Rrboh D表達(dá)明顯低于對(duì)照,At ICL、NADP-ME1、At ADH1表達(dá)顯著高于對(duì)照。EMAS(凝膠阻滯實(shí)驗(yàn))分析發(fā)現(xiàn),ZmNTL1-△TM蛋白能夠與At Rboh D啟動(dòng)子的4個(gè)位點(diǎn)結(jié)合,表明ZmNTL1直接調(diào)控At Rboh D的表達(dá)。發(fā)現(xiàn)在水淹下,擬南芥rbohd(敲除系)生長(zhǎng)受到抑制,而35S::At Rrboh D生長(zhǎng)優(yōu)于對(duì)照,表明At Rrboh D參與了擬南芥對(duì)水淹脅迫的調(diào)控。將At Rboh D的過(guò)表達(dá)載體轉(zhuǎn)入35S::ZmNTL1-△TM中,35S::ZmNTL1-△TM對(duì)水淹的敏感性得到恢復(fù),從遺傳上證明了ZmNTL1和At Rboh D的直接調(diào)控關(guān)系。綜上,ZmNTL1作為一個(gè)水淹脅迫的負(fù)調(diào)控因子,可通過(guò)直接下調(diào)At Rboh D表達(dá)來(lái)降低對(duì)水淹的耐受性。(9)ZmNTL2在調(diào)控植物木質(zhì)素合成中的作用研究35S::ZmNTL2和35S::ZmNTL1-△TM轉(zhuǎn)基因擬南芥生長(zhǎng)7周時(shí),莖的硬度明顯優(yōu)于野生型。甲苯胺藍(lán)和間苯三酚染色發(fā)現(xiàn),與野生型相比,過(guò)表達(dá)系植株莖木質(zhì)部中被染色的細(xì)胞數(shù)明顯增多,且著色深,表明木質(zhì)素分布范圍和含量均高于對(duì)照。原位雜交結(jié)果表明,ZmNTL2主要在木質(zhì)部表達(dá)。Real-time PCR分析表明,木質(zhì)素合成的關(guān)鍵基因PAL、C4H、HCT、CAD、CCR、C3H、COMT、F5H在35S::ZmNTL2過(guò)表達(dá)系和35S::ZmNTL1-△TM過(guò)表達(dá)系中明顯上調(diào)。綜上,ZmNTL2可能通過(guò)上調(diào)木質(zhì)素合成相關(guān)基因表達(dá)來(lái)提高植株莖的木質(zhì)化程度�？傊�,玉米ZmNTL家族成員基因在植物發(fā)育和響應(yīng)非生物脅迫中起著重要作用,挖掘ZmNTLs基因的功能對(duì)解析玉米生長(zhǎng)發(fā)育及耐逆性作用機(jī)制有重要意義。
[Abstract]:Transcription factors play an important role in regulating plant development and the expression of anti inverse function genes. NAC transcription factors are specific regulatory factors of plants, which play important roles in plant organ development, morphogenesis, organ separation, leaf senescence and abiotic stress response. Membrane binding transcription factors are a class of transcription factors. A special transcription factor, usually in the form of membrane binding that exists in the membrane structure of the cell (cell membrane, endoplasmic reticulum or nuclear membrane) and is in an inactive state. After being stimulated by external signals, it is released from the membrane through the proteolysis mechanism and transformed into active form into the nucleus. The functional study of NAC membrane binding transcription factor (ZmNTLs) has not yet been reported. In this study, 7 ZmNTLs genes were unearthed from the maize genome. Their protein structure characteristics, phylogenetic tree, subcellular location, transcriptional activation activity, tissue specific expression, and characteristics of abiotic stress and hormone response were studied. 35S:: ZmNTL1,35S:: 35S:: ZmNTL1,35S:: ZmNTL2 and 35S:: ZmNTL5 pure lines, and the role of ZmNTL1 and -2 in regulating water flooding stress response and lignin synthesis in Arabidopsis, respectively. The main research process and experimental results include: (1) ZmNTLs sequence of transcription factor in corn NAC membrane, expression characteristics analysis using bioinformatics method, from 216 corn NAC to NAC 7 members with transmembrane domain were selected and identified as NAC membrane binding transcription factor (ZmNTL), and named as ZmNTL1-7. sequence alignment. All maize NTLs N- ends have typical NAC domain, and all C- ends have 1 transmembrane spiral domains. Amino acid sequence alignment and 7 ZmNTLs are divided into 4 groups (packages). Including 3 gene pairs, ZmNTL1/5, ZmNTL2/3, ZmNTL6/7 and ZmNTL4. (2) ZmNTLs evolution analysis using MEGA6.0 to 18 NTLs proteins from Arabidopsis, 5 NTLs proteins of rice, 8 NTLs protein of millet and 7 NTL proteins of maize, the results showed that the above 38 membrane binding transcription factors were divided into 5 subgroups, ZmNT Ls is distributed in the 4 subgroups of I-IV, and ZmNTL2/ZmNTL3 and At NTL4 are located in group I, ZmNTL4 and At NTL7 are in group II. The analysis showed that the conservative motif of maize NTLs compared the evolutionary relationship between.ZmNTLs and At NACs, Os NACs, and found that ZmNTLs and some NAC transcriptional factors in Arabidopsis have closer evolutionary relationships, such as ZmNTL1 and ANAC086, ANAC045, etc., which may have similar functions to some Arabidopsis. 3) ZmNTLs subcellular localization and transcriptional activation activity analysis using maize B73 inbred line as material, cloned 7 ZmNTLs genes CDS, constructed their GFP transient expression vector, transformed them into Arabidopsis protoplasts, and found ZmNTL1-5 and 7 located on the cytoplasmic membrane; ZmNTL6 was located on the endoplasmic reticulum. Yeast activation active fraction was determined. It was found that all ZmNTLs had transcriptional activation activity. Quantitative detection of beta galactosidase reporter gene showed that ZmNTL2 transcriptional activation activity was the highest, followed by ZmNTL6; ZmNTL4 and ZmNTL7 were relatively weak. (4) Real-time PCR analysis of ZmNTLs's spatio-temporal expression pattern showed that ZmNTL1-7 expression had obvious tissue specific.ZmNTL1, -2 in roots, stems and leaves. The expression of ZmNTL1 was the highest in the leaves, the expression of ZmNTL2 in stem was the highest, and ZmNTL3-7 was mainly expressed in the stem. (5) ZmNTLs was used for abiotic stress (drought, high salt, low temperature, H2O2) and hormone (ABA, BR, GA, 2,4-D, Me JA, KT) in the three leaf period maize seedlings. R analysis showed that ZmNTL1-7 showed diversity in the expression patterns of abiotic stress and hormone response. Under the stress of ABA and H2O2, ZmNTL1-7 was significantly up-regulated in the root and stem, and the expression pattern was similar, while ZmNTL1-7 was significantly down regulated in the stem under PEG treatment; ZmNTL1-7 was induced to be up expression by ABA, Me JA, BR. The response to ABA and Me JA was most intense; GA treatment was the weakest, ZmNTL1, -4, -6 had a small amount of induction, ZmNTL7 was suppressed, ZmNTL2, -3, -5 had no obvious changes. The homozygous seeds of transgenic Arabidopsis were obtained. It was found that the length of the main root and the growth potential of the transgenic Arabidopsis were not different from those of the wild type. (7) the response of the overexpression line of ZmNTL Arabidopsis to ZmNTL1, -2, and -5 to remove the transmembrane domain (delta TM) of the transgenic Arabidopsis overexpressed line (Col-0) On the 1/2MS solid medium, the seeds were transferred to the medium containing 0.5mM, 1mM, 1.5mM and 2mM H2O2 on the medium of 1cm, respectively. The results showed that the transgenic lines could significantly alleviate the inhibition of hydrogen peroxide, and the three ZmNTLs genes were responsive to hydrogen peroxide stress. (8) the effect of ZmNTL1 on water flooding stress Under the condition of soil water saturation, the growth state of 35S:: ZmNTL1 transgenic Arabidopsis was not significantly different from that of the control. The growth state of 35S:: ZmNTL1- Delta TM was obviously weaker than that of the control. The growth retardation, the dwarf plant and the curl of leaves showed that ZmNTL1 decreased the tolerance.Real-time PCR analysis of Arabidopsis to water flooding stress, which showed that the water flooded, under water flooding, 35S:: the expression of At Rrboh D in ZmNTL1- Delta TM was significantly lower than that of the control. The expression of At ICL, NADP-ME1, At ADH1 was significantly higher than that of the control.EMAS (gel block experiment). The growth of 35S:: At Rrboh D is better than that of the control, which indicates that At Rrboh D participates in the regulation of water flooding stress in Arabidopsis thaliana. The over expression vector of At Rboh D is transferred to 35S:: ZmNTL1- Delta, the sensitivity to water flooding is restored. For a negative regulator of water flooding stress, the tolerance to water flooding can be reduced by direct downregulation of At Rboh D. (9) the effect of ZmNTL2 on the regulation of lignin synthesis in plants: 35S:: ZmNTL2 and 35S:: ZmNTL1- Delta and TM transgenic Arabidopsis thaliana growth at 7 weeks, the stem hardness is better than that of wild type. Toluidine blue and benzol three phenol are dyed. Compared with the wild type, the number of stained cells in the xylem of the overexpressed plant increased obviously, and the coloring was deep, indicating that the distribution and content of lignin were higher than those of the control. The results of in situ hybridization showed that ZmNTL2 was mainly expressed in xylem by.Real-time PCR analysis, which indicated that the key genes of lignin synthesis, PAL, C4H, HCT, CAD, CCR, C3H, COMT, F5H. In 35S:: ZmNTL2 overexpression line and 35S:: ZmNTL1- Delta TM overexpression line obviously up-regulated. To sum up, ZmNTL2 may improve the lignification degree of plant stem by up regulation of lignin related gene expression. In a word, the gene of maize ZmNTL family members plays an important role in plant development and response to abiotic coercion, mining the function of ZmNTLs gene. It is important to understand the mechanism of maize growth and development and stress tolerance.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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本文編號(hào)：1940520